The 30/32-kDa complex of major secretory proteins are among the most important and intensively studied proteins of Mycobacterium tuberculosis. The proteins have been demonstrated to be immunoprotective and to play a central role in the physiology of the mycobacterium. In this study, we present a series of novel insights into this key protein complex arising out of a combination of genetic, biochemical, and immunocytochemical analyses. Our genetic analyses (i) indicate that the genes are arranged as separate transcription units, (ii) demonstrate that the mature 30-kDa protein of M. tuberculosis differs from the corresponding 30-kDa proteins of two strains of Mycobacterium bovis BCG by only 1 and 5 amino acids, (iii) suggest that expression of the proteins is regulated at the transcriptional level, and (iv) map the transcriptional start site of the 30-kDa protein gene. Our biochemical analyses provide evidence that (i) the 30-kDa protein and the two 32-kDa proteins (i.e., 32A and 32B) are secreted at a ratio of approximately 3:2:1, respectively, (ii) the proteins exist as monomers, (iii) the proteins are not posttranslationally modified by the addition of carbohydrates and lipids, (iv) the 30-kDa and 32A proteins contain one disulfide bridge, and (v) high-level expression and leader peptide processing are achievable in Escherichia coli. Our immunocytochemical analyses demonstrate that the 30/32-kDa complex is expressed in human monocytes and that the proteins are localized to the phagosomal space and the mycobacterial cell wall. These analyses fill important gaps in our knowledge of this critical protein complex of M. tuberculosis and, at the same time, raise new and fundamental questions regarding regulatory mechanisms that control coordinate expression of the proteins at a fixed ratio.